Techniques for specifically amplifying a target nucleic acid sequence are very important for molecular biology research and clinical applications (e.g. genetic testing). An amplified product obtained by a nucleic acid amplification technique can be specifically detected, for example, by immobilizing a target sequence-containing nucleic acid fragment on a solid phase. In this method, the target nucleic acid is specifically immobilized on the solid phase, and non-specific nucleic acid sequences can then be easily removed by washing or the like. Thus, detection specificity can be enhanced.
In the method, the target nucleic acid may be captured on the solid phase by using an antigen-antibody or ligand-receptor pair capable of specifically binding together. For example, Non Patent Literature 1 discloses a method for detecting a PCR product amplified using a primer pair including a primer terminally modified with biotin and another primer modified with a fluorescent substance. This method includes contacting the PCR product with a streptavidin-agarose solid phase, forming a streptavidin-biotin complex to bond the PCR product to the solid phase, and measuring fluorescence of the complex, whereby the target amplified product can be detected.
Unfortunately, the number of antigen-antibody or ligand-receptor combinations usable for labeling is limited, which makes it substantially difficult to detect multiple target nucleic acids at one time. Another problem is the cost: fluorescently labeled nucleic acids are expensive.
Another method for capturing a target nucleic acid on a solid phase is to immobilize, on the solid phase, an oligonucleotide probe containing a sequence complementary to the target nucleic acid, and then indirectly immobilize the target nucleic acid on the solid phase through hybridization of the target nucleic acid and the probe. In this method, the intensity of a signal generated upon hybridization is detected. Such a nucleic acid analysis allows multiple target sequences to be analyzed at one time by using varied probe sequences.
In general, in order to hybridize an immobilized probe and a target nucleic acid on a solid phase, the double-stranded nucleic acid amplified by PCR needs to be denatured into single strands by heat treatment. Unfortunately, the heat treatment is troublesome and also has the problem of reduced hybridization efficiency due to reannealing. Another problem is that single-stranded DNA tends to easily curl into balls and is thus inferior in detection sensitivity. Patent Literature 1 discloses a method for amplifying a single-stranded nucleic acid using nuclease treatment without heat treatment; however, it is also troublesome to operate and has the problem of curling of single strands into balls.
The chromatography-based method disclosed in Patent Literature 2 is easy to operate and allows rapid and simple detection of target nucleic acids as compared to other nucleic acid detection methods. This is a gene detection method that includes the steps of: sampling genes from a cell, virus or bacterium, fragmenting the randomly sampled genes, and detecting a target gene, wherein these steps are continuously performed on a single device for detecting genes by transferring a liquid sample containing the randomly sampled genes or fragments thereof by capillary action. This method allows assessment of the presence of a target gene and identification of the type of gene. Still, Patent Literature 2 employs NASBA to amplify single-stranded nucleic acids. It has the problems associated with the use of single-stranded nucleic acids as described above.
In order to solve the above problems, Patent Literatures 3 and 4 propose the use of a primer region having on the 5′ side a non-natural nucleic acid tag, a hairpin structure or a pseudoknot structure for inhibiting nucleic acid synthesis by DNA polymerase to leave a single-stranded region in one strand of a double-stranded nucleic acid after PCR reactions. This technique is advantageous in that an amplified double-stranded DNA product having a hybridizable single-stranded region at one end can be produced by only performing PCR reactions using such a special primer. However, it requires detection using fluorescent labeling or surface plasmon resonance imaging, which requires expensive special equipment. Thus, there are problems with speed and simplicity.